Chimeragenesis: a method for generating, selecting, and phenotyping gene variant libraries in yeast
Authors: Cory Weller; Meru Sadhu
Affiliation: National Human Genome Research Institute, NIH, Bethesda, MD
Keywords: Complex traits
Thorough study of genetic diseases requires understanding how amino acid substitutions influence phenotype. Manually engineering individual substitutions can provide insight into small numbers of variants, but assessing many variants requires high-throughput methods. Here, we describe a method that improves upon deep mutational scanning by allowing for high-throughput selection, sequencing, and phenotyping of variants within a single pool. First, we prepare a plasmid library via assembly of variant-generating repair templates amplified from a cost-effective synthesized oligonucleotide pool. Each plasmid, when transformed into a yeast cell expressing Cas9, directs the generation of a specific predetermined variant. We transform our plasmid library into a yeast strain that possesses two copies of our gene of interest separated by a counter-selectable marker. A given repair template induces a large-scale deletion between the gene copies, removing the counter-selectable marker while recapitulating a normal-length gene of interest. Variants can be generated either by virtue of the two gene copies exhibiting amino acid substitutions (thereby generating a recombinant gene) or by introducing novel codons via the repair template. This deletion-based method greatly simplifies CRISPR-Cas9 editing by requiring a single, shared guide RNA for the entire variant library. It also allows for simple selection of mutants and facilitates pooled tracking of variant abundance (inferred from abundance of repair templates in pooled short-read sequencing). This technique will greatly accelerate the generation and assessment of variant effects, providing insight into protein function and the genetic underpinnings of disease.